Intra-articular adipose tissue is an important structural tissue within joints and is increasingly recognized as a critical mediator of inflammation. The extrinsic factors that regulate intra-articular adipose tissue inflammation are unknown. Identifying such factors and the molecular mechanisms by which they impact joint inflammation will advance the development of new treatments for joint injury and musculoskeletal disability. The applicant's long-term goal is to develop therapies to treat and prevent obesity-associated osteoarthritis by identifying biomechanical and metabolic factors that promote the resolution of joint inflammation. This application focuses on the mechanobiology of inflammatory signaling in intra-articular fat pads. The objective here is to identify the effect of joint loading on the expression of inflammatory adipokines within the infrapatellar fat pad (IFP) and to determine whether adipose tissue macrophages (ATMs) mediate this response. The central hypothesis is that physiologic joint loading due to exercise induces resident ATMs to initiate a pro-fibrotic response that restricts adipocyte hypertrophy and thereby attenuates pro-inflammatory adipokine expression. The hypothesis and focus on ATMs as mechano-sensitive pro-fibrotic mediators in the IFP is based on a comprehensive literature review and preliminary data generated in the applicant's laboratory. These findings indicate that compared to subcutaneous fat, the IFP has an elevated expression of pro-fibrotic growth factors, fibronectin, and cytokines. Preliminary wheel running studies also show that the IFP is a dynamic structural tissue that increases extracellular matrix deposition in response to exercise. Guided by these data, the project will test the hypothesis using two specific aims: 1) Determine the time-course of in vivo and ex vivo biomechanical stimulation on mediators of macrophage infiltration, polarization, and IFP fibrosis; and 2) Determine the requirement of resident ATMs on biomechanically-stimulated IFP fibrosis and adipokine expression. A well- established voluntary wheel running mouse model will be used to evaluate the effect of increased IFP mechanical stimulation on gene, protein, and cellular outcome measures. The applicant's lab will also utilize an ex vivo tissue compression system for conducting compressive loading experiments on rat IFP samples. Under the second aim, resident ATMs will be depleted in these in vivo and ex vivo models with liposomal clodronate to determine if ATMs mediate the effect of biomechanical stimulation on IFP fibrosis and adipokine expression. The proposed research is significant because it is expected that biomechanical stimulation will greatly contribute to the paracrine inflammatory signaling function of intra-articular fat pads. Ultimately, such knowledge is expected to lead to the development of novel therapeutic targets for pre-clinical testing in the prevention or treatment of diseases involving musculoskeletal inflammation and physical disability.